VSAD1 Involved In The Regulation Of Plant Resistance To Verticillium Wilt

Verticillium wilt is a kind of plant vascular disease, it is caused by soil-borne fungi--Verticillium dahliae.This disease is distributed in every cotton-producingarea around the world.When cotton infected by V.d, the leaf will become chlorisis and the plant died finally.As the pathogenic mechanism of bacteria and mechanism of plant resistance is stillunclear,and lack of high-efficient resistant materials,these characteristics make it hard to the research and prevention of Verticillium wilt.Now finding resistant gene through molecular method is practicalway.Because the genome of cotton is large and complex,it is hard to find resistant gene.Arabidopsis is a model plant, it is the host of Verticillium dahlie,and it has a clear genetic background, it can be used as the study material for the mechanism of plant resistance to Verticillium dahliae.Based on theinteraction system for the Arabidopsis and Verticilliumdahliae, our group screen Arabidopsis EMS mutants library. This study appraisal a batchof Arabidopsis mutants, which are sensitive to Verticilliumdahliae,vsad1-1 is one of those mutants.When infected by Verticillium dahlia, the leaf of vsad1-1 become chlorisis in a fewdays and the concentrition of anthocyanin is low, the freshweight and content ofchlorophyll decreased.In previous work, our group have proved that the VSAD1 is a recessive gene.Through map based cloning,we finally know that the gene is located on upstream of chromosome ?. vsad1 is a mutant of At5g13930.Gene mutation leads the 184' amino acids of this protein change from glutamic acid(E) to lysine(K).At the same period, we also screened another mutant vsad1-2, it's a allelic mutant of vsad1-1,gene mutation leads the 49' amino acids change from threonine(Thr) to lysine(Ile).In order to verify the correctness of the mutation site,we get the mutanttt4-2 from SALK to repeat phenotype under V.d. tt4-2 is a mutant of VSAD1 In MS,the phenotype of tt4-2 and vsad1-1 is almost the same: compared with the wild type, cotyledons of the two mutants etiolated early,have low anthocyanin accumulation, bolt early.And the content of chlorophyll in the leave decreased heavily.When the two mutants infected by Verticillium dahlia in the soil, the death rate increased by 15%.Through transforming the construction of VSAD1 over-expression vector into vsad1-1, theover-expression linesrestored the ability of anthocyanin accumulation in different levels.These result indicate that: vsad1 is a mutant generated by the mutation of At5g13930.In order to analysis the expression features of VSAD1, we extract the RNA from the leaf of Arabidopsis which is infected by V.d. RT-PCR showed that the gene expression of VSAD1 is induced by Verticilliumdahliae.When infected, the expression increased significantly. VSAD1:GUS staining showed that, under normal circumstances, the whole plant were slightly stained, while GUS staining deepen after the plant was infected by Verticillium dahliae,these indicate that pathogen would induce the expression of this gene.GUS enzyme activity also increased significantly by 43.6% when the plant was treated by Verticillium dahlia.In order to analysis the relationship between sensitivity of vsad1-1 to Verticillium dahliaand the content of Verticillium dahliain plant.According to the literature report, we design primers whcih is specific to Beta tubulin of Verticilliumdahliae to quantify the content of Verticilliumdahliae in plant by QRT-PCR. The result indicate that there are more Verticillium dahliae in vsad1-1 than WT.Previous study has proved that VSAD1 involed in the synthesis of anthocyanin, so we design another experiment to show that the common grape seed proanthocyanidins mixture could inhibit the growth of Verticillium dahliae.With the increase of concentration, Verticillium dahliae.growth arrest. At the same time, we extract the anthocyanin from the leaf of Arabidopsis.And we use this extractiveto repeat inhibition test. The result is as same as the grape seed proanthocyanidins.The anthocyanidins of Arabidopsis could inhibit the growth of Verticillium dahliae.These result mean that there has some relationship between susceptibility of vsad1-1 and the content of Verticillium dahliae in the plant.In order to testify whether VSAD1 invovled in cottonresistance to Verticillium dahlia, we find the homologus gene –GhCHSin cottonthrough BLAST, which has been published in NCBI. The amino acid sequence similarity between VSAD1 and GhCHSis 84%.In the early stage of infection,the expression of cotton's homologous gene--GhCHS is upregulated. When the expression of GhCHS is downregulatedthrough VIGS, cotton become more sensitive to Verticillium dahliae and.the diease index lift up.about 8%.Above all,VSAD1 involved in the regulation of plant resistance to Verticillium wilt.